Detection of canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 overexpressing skin papillomas of the dog using the polymerase chain reaction and non-radioactive in situ hybridization

Nineteen cutaneous and mucocutaneous papillomas, as well as 29 oral and 25 non-oral squamous cell carcinomas of dogs were analyzed immunohistologically for the presence of papillomavirus (PV)-antigens. Canine oral papillomavirus (COPV)-DNA was detected in formalin-fixed, paraffin-embedded tissues by polymerase chain reaction (PCR) and non-radioactive in situ hybridization (ISH). Furthermore, the expression of the tumor suppressor protein p53 was investigated. PV-antigens were detectable in more than 50% of the oral and cutaneous papillomas, while no PV-antigens could be demonstrated in venereal papillomas. One squamous cell carcinoma was PV-antigen positive. Only two cutaneous papillomas of the head showed a strong p53-specific immunostaining, while overexpressed p53 was detectable in approximately 35% of all squamous cell carcinomas. It was possible to amplify fragments of the E6, E7 and L1 gene by polymerase chain reaction (PCR) from five of eight oral and from five of eight cutaneous papillomas as well as from three oral squamous cell carcinomas. Nine of 10 papillomas showed a strong nucleus-associated hybridization signal typical for COPV-DNA. In three squamous cell carcinomas COPV-DNA was located in nests of the epithelial tumor cells surrounding ‘horn pearls' or disseminated in the carcinoma tissue. These observations support the view that COPV may also induce non-oral papillomas in the dog and confirm the opinion that a progression of viral papillomas into carcinomas in dogs may occur.     

Papillomas and carcinomas associated with a papillomavirus in European harvest mice (Micromys minutus)

Papillomaviruses, group-specific papillomavirus antigens, or extrachromosomal papillomavirus DNA were detected in cutaneous, mucocutaneous, and pulmonary tumors affecting a colony of European harvest mice (Micromys minutus). Skin lesions were classified as acanthomatous hyperplasia, epidermal inclusion cysts, squamous papillomas, inverted papillomas, trichoepitheliomas, and sebaceous carcinomas. Cutaneous horns (hyperkeratotic papillomas) were on mucocutaneous junctions of one animal. One mouse, with a cutaneous sebaceous carcinoma, had multiple pulmonary keratinaceous cysts. Papillomavirus antigens, detected by the avidin-biotin technique, were in 20 of 31 lesions tested. In contrast, by Southern blot hybridization all 28 lesions tested contained papillomavirus DNA. Papillomavirus DNA was demonstrated in two of ten benign cutaneous lesions by in situ hybridization.     

Early Changes in Pigmented Macrophages in Head Kidney of Channel Catfish Infected with Aeromonas hydrophila

Abstract

Channel catfish Ictalurus punctatus with scarified skin were exposed to the bacterial pathogen Aeromonas hydrophila. Systemic infections developed in 80% of the exposed fish, and the remaining exposed fish had infections limited to the cutaneous lesion. Skin of control fish was injured in the same manner as for the exposed fish, but control fish were not exposed to A. hydrophila nor was A. hydrophila recovered from them. None of the fish died during the 3-d experiment, and gross lesions in head kidneys, including changes in the size or relative weight, were not found in infected fish. Macrophage aggregates in head kidney of systemically infected fish increased 68% in mean area, 28% in number, and 111% in total relative volume. Volume of lipofuscin, the predominate pigment found in channel catfish head kidney, in systemically infected fish was about twice that of control fish. Volume of hemosiderin was about four times as great and macrophages containing hemosiderin were about three times as numerous in systemically infected fish as in control fish. The amount of melanin in macrophages did not change in infected fish. Significant differences in macrophage aggregates and pigments were not found between controls and fish with only superficial infections. Microscopic lesions, other than changes in macrophage aggregates and pigments, were not found in the head kidney. Increases in macrophage aggregates, lipofuscin, and hemosiderin in head kidney of channel catfish were useful for quantification of injury caused by a systemic bacterial infection.     

Digital papillomatosis in a confined Beagle.

Papillomavirus-induced papillomas were diagnosed on multiple digits of all 4 feet of a young Beagle. No other cutaneous or oral involvement was identified. Papillomavirus antigen was confirmed by immunoperoxidase localization within keratinocyte nuclei. In addition to the typical basophilic intranuclear inclusions associated with papillomavirus infections, keratinocytes within the papillomas contained large, eosinophilic cytoplasmic inclusions that previously have been described in a Boxer with cutaneous lesions associated with a papillomavirus infection. The papillomas in this Beagle regressed completely within 2 months of the initial diagnosis.     

Canine inverted papillomas associated with DNA of four different papillomaviruses

Inverted papillomas are uncommon papillomavirus (PV)‐induced canine skin lesions. They consist of cup‐ to dome‐shaped dermal nodules with a central pore filled with keratin. Histologically they are characterized by endophytic projections of the epidermis extending into dermis. Cytopathic effects of PVs infection include the presence of clumped keratohyalin granules, koilocytes and intranuclear inclusion bodies. Different DNA hybridization studies carried out with a canine oral papillomavirus (COPV) probe suggested that a different PV than COPV might cause these lesions. Canine papillomavirus 2 (CPV2) was discovered a few years ago in inverted papillomas of immunosuppressed beagles. Two other cases, presenting with distinct clinical and histological features have also been described. This study was carried out on four dogs with clinical and histological signs of inverted papillomas. Molecular biological analyses confirmed that PV DNA was present in all four lesions but demonstrated that the sequences in each case were different. One corresponded to COPV, the second to CPV2, and the third and fourth to unknown PVs. These findings suggest that inverted papillomas are not caused by one single PV type. Similar observations have also been made in human medicine.     

Epidermal Tumors on Rainbow Smelt and Atlantic Salmon from Nova Scotia

Abstract

Epidermal tumors on rainbow smelt Osmerus mordax are described in detail for the first time. These tumors apparently progress from small preneoplastic lesions to papillomas, and to carcinomas on some males. These tumors and similar tumors on Atlantic salmon Salmo salar have not been previously reported from Canada. Tumors were found on rainbow smelt mainly in freshwater during the spring spawning run and on Atlantic salmon grilse in freshwater in October, These tumors varied in size and number on each fish. The smaller growths were usually translucent and whitish, with a smooth surface. Some larger tumors were papillomatous and appeared reddened because of increased vascularization. Preneoplastic lesions and papillomas had a thickened epidermis with an intact basement membrane, and most of the cells were enlarged. fn papillomas of rainbow smelt, tumor cells that were not close to papillae were vacuolated, and necrotic regions were present. Carcinomas on the heads of male rainbow smelt had regions of pleomorphic cells which invaded the subepithelial tissues and penetrated the cartilage of the mandible of two specimens found in brackish and seawater. Intranuclear basophilic inclusions suggestive of a viral agent were present in the tumors of both rainbow smelt and Atlantic salmon.     

Treatment factors influencing the use of recombinant platelet-derived growth factor (Regranex) for head and lateral line erosion syndrome in ocean surgeonfish (Acanthurus bahianus)

The clinical efficacy of becaplermin (Regranex, Ortho-McNeil Pharmaceutical, Inc., Raritan, New Jersey 08869) on head and lateral line erosion syndrome (HLLES) in ocean surgeonfish (Acanthurus bahianus) was examined in a two-part study to determine suitable application frequencies of the drug and the effects of the environment on outcome of treatments. In the first part of the study, 12 ocean surgeonfish with prior HLLES were equally divided into three treatment groups, varying in application frequency of becaplermin: 1) one application, 2) three applications every 3 wk, and 3) no applications. After 9 wk, it was determined through photograph and computer analysis that fish treated with becaplermin did heal significantly more than the fish that were not treated. No significant difference was found when comparing the two treatment regimes; therefore, a one-time treatment of becaplermin was just as effective as the three-time application, reducing cost of the treatment, staff labor, and stress to the animals. In the second part of the study, the effect of the water environment on HLLES development was documented before and after a one-time treatment of becaplermin. When fish were treated and placed into a system known to cause HLLES, the becaplermin treatment had no effect on reducing the HLLES progression, whereas treated fish that were placed into a system that did not cause HLLES showed a significant decrease in erosion after the treatment. Therefore, treating fish being placed into a HLLES-causing system serves no beneficial purpose to healing HLLES.     

Langerhans' cells in equine cutaneous papillomas and normal skin.

Langerhans' cells (LC) were investigated immunohistochemically and electron microscopically in normal equine epidermis and 133 equine cutaneous papillomas experimentally induced in five 2-year-old Thoroughbred horses. Class II major histocompatibility complex antigen-positive dendritic LC were found in the normal epidermis and ultrastructurally had the characteristic Birbeck's granules. In the developing phase of the papillomas, LC were significantly decreased in number and size, indicative of a hypofunctional state. In the regressing phase of the papillomas, LC were markedly increased in number, especially at the epidermis-dermis junction. LC with long dendrites were rich in cytoplasm with well-developed cytoplasmic organelles, including Golgi apparatus, lysosomes, Birbeck's granules, and multivesicular bodies. These LC were hyperfunctional. An infiltration of many T lymphocytes was also observed at the epidermis-dermis junction.     

Papillomatosis of the bovine teat (mammary papilla)

A 4th of 667 cattle examined at a Wisconsin abattoir had teat papillomas. Excised teat papillomas were sorted by gross morphologic characteristics into 3 groups: (i) atypical filiform, (ii) atypical flat, and (iii) typical fibropapilloma. Bovine papilloma virus capsid antigen was detected in thin-section slides of the 3 groups of teat papillomas by peroxidase-antiperoxidase assay. The bovine papilloma virus involved with the atypical papillomas could not be characterized by molecular hybridization, because enough pure virus could not be harvested. Homogenates of the 3 groups of teat papillomas were inoculated on 2 ponies and 4 calves. Typical fibropapillomas were produced on the 4 calves, and fibromas, on the 2 ponies. Atypical papillomas were produced only in 2 heifers.     

Resolution of persistent oral papillomatosis in a dog after treatment with a recombinant canine oral papillomavirus vaccine

This report describes a 16‐month‐old female, otherwise seemingly healthy, Siberian husky dog with severe oral papillomatosis that did not regress spontaneously and was refractory to surgical treatment over a 6‐month period. Regression of the papillomas was achieved by administering a series of experimental vaccinations starting at the time of the last surgery. The vaccine consisted of systemically administered canine oral papillomavirus major coat protein L1 that has been shown to self‐assemble into virus‐like particles. They cause a humoral response that has been shown to prevent the onset and development of papillomas. In this case, however, following unsuccessful surgical treatment, the vaccine acted therapeutically, causing the papillomas that had regrown to shrink. No side‐effects were noted.     

Investigation of parrot papillomavirus in cloacal and oral papillomas of psittacine birds

Cloacal and oral papillomas from 27 psittacine birds of various species were examined for the presence of parrot papillomavirus by DNA in situ hybridization, DNA in situ polymerase chain reaction, and nested polymerase chain reaction. Parrot papillomavirus was detected in one oral papilloma from an African grey parrot by all three techniques. In addition, rare basophilic intranuclear inclusions were observed by light microscopy in tissue sections of the oral papilloma from this parrot. The remaining lesions were negative for parrot papillomavirus DNA. This study suggests that parrot papillomavirus may be involved in the development of papillomas in African grey parrots, but apparently is not responsible for development of similar lesions in unrelated species of psittacine birds.     

奶牛头状瘤治疗的免疫研究

采用外科手术部分摘除法、液氮冷冻疗法和瘤体灭活疫苗疗法分别治疗奶牛乳头状瘤各６例，并选用对流免疫电泳和非特异性醋酸萘酯法，结合免疫指标（抗体水平和Ｔ淋巴细胞值）的定期测定，初效，其中以免疫疗法效果最好，保护期最长，是一种实用的治疗方法。奶牛乳头部乳头状瘤自愈，初期是以Ｔ淋巴细胞为主导的细胞免疫为主，后期以抗体介导的体液免疫应答为主导的综合结果     

Evidence for papillomaviruses in ocular lesions in cattle

Negatively stained preparations of various ocular lesions, generally considered to be precursors to bovine ocular squamous cell carcinoma, were subjected to electron microscopic examination for viruses. Lesions examined included five conjunctival plaques, an acanthotic lesion from an eyelid, 15 conjunctival papillomas, two papillomas of the eyelid and two keratinised elongated proliferative lesions of the eyelid (cutaneous horns). Eight lesions contained particles that resembled papillomaviruses. These consisted of a conjunctival plaque, five conjunctival papillomas, a papilloma of the eyelid and one of the samples of cutaneous horn. The particles were present in small numbers and were found only after prolonged search. The finding of these particles suggests that papillomavirus may be involved in the aetiology of bovine ocular squamous cell carcinoma.     

Experimental transmission of bovine papilloma virus (BPV) extracted from morphologically distinct teat and cutaneous lesions and the effects of inoculation of BPV transformed fetal bovine cells.

Bovine papilloma virus (BPV) was extracted from five cattle each affected with only one of five morphologically distinct lesion types. When inoculated into experimental calves either by scarification or intradermal injection, the BPV extracts produced lesions macroscopically and microscopically similar to those from which individual extracts were made. Fetal bovine cells, transformed in vitro with BPV, failed to produce fibromas, fibropapillomas or papillomas when inoculated into experimental calves. When calves inoculated with virus or BPV transformed cells were challenged with the five original BPV extracts, a differential immunity was demonstrated, while control calves were susceptible to all extracts. Post mortem examination revealed the presence of upper alimentary tract papillomas in three of eight calves forming one group. These results suggest that different strains of BVP, causing morphologically separable lesion types, exist. There may be additional BPV variants causing fibropapillomas of the teat and anogenital regions of cattle. The inoculation of BPV transformed fetal bovine cells conferred a relative immunity to later challenge with some but not all BPV extracts.     

Graft-versus-host reaction (GVHR) in clonal amago salmon,Oncorhynchus rhodurus

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.     

Cellular immunity in salmonids infected with the microsporidial parasite Loma salmonae or exposed to non-viable spores

Following a per os challenge of naive rainbow trout with live spores of Loma salmonae, head kidney mononuclear cells (MNC) in culture were able to proliferate in response to crude soluble parasite extract or intact dead spores. A significant response was seen by week 2 post-exposure and a maximum response developed by week 6 or 8, respectively. During this initial challenge, spore filled cysts developed on the gills of challenged fish, and the cysts ruptured by week 12 as is typical for microsporidial gill disease of salmonids (MGDS). Two weeks following this, fish were re-challenged with live spores, and in these fish an enhanced in vitro proliferative response of MNC was immediately apparent, and spore filled cysts did not develop. In contrast, when naive trout were given dead spores by intraperitoneal injection, the most pronounced proliferative responses of MNC developed earlier (week 2 PE) and the response was greater when cells were incubated in vitro with dead spores rather than with crude soluble extract. When these fish were re-challenged per os with live spores, a heightened proliferation in MNC was observed 4 weeks after this exposure and the fish likewise resisted development of xenomas. In fish infected orally or injected intraperitoneally with spores, a marked increase in the response to the mitogen concanavalin A was seen for 22 weeks post-exposure when compared to controls not receiving any spores.     

Immunosuppressive effect of infectious pancreatic necrosis virus on rainbow trout (Oncorhynchus mykiss)

The infectivity of infectious pancreatic necrosis virus (IPNV) for rainbow trout (Oncorhynchus mykiss) mononuclear leukocyte subpopulations was investigated to determine the mechanisms of immunosuppression caused by the virus. IPNV was recovered from nylon wool-adherent, surface immunoglobulin (Ig)-positive leukocytes of head kidney, spleen and peripheral blood collected from virus-inoculated fish with higher titers than non-adherent, Ig-negative cells. Non-adherent cell population showed mitogenic response to phytohemagglutinin and concanavalin A but not to lipopolysaccharide. Conversely, the responses of adherent cells to these mitogens were weak. Mitogenic response and non-specific cytotoxicity of head kidney leukocytes significantly decreased by the inoculation of fish with the virus. These results suggest that the suppression of immune responses is involved in the establishment of carrier state in fish after infection with IPNV.     

Cutaneous inverted papillomas in dogs

Inverted papillomas of the skin occurred in five dogs. Lesions were 1-2 cm, circumscribed, flask-like structures below the level of the surrounding normal skin. Walls of the structures consisted of hyperplastic epidermis, forming thin papillary projections on thin fibrovascular stalks. Cells in the stratum granulosum had clear cytoplasm, numerous keratohyalin-like granules of various sizes, and poorly defined intranuclear inclusions. These cells stained positively for papillomavirus group-specific antigens by both the peroxidase-antiperoxidase and avidin-biotin methods. Virions with a mean diameter of 35.7 nm were present within nuclei in cells of the stratum granulosum when examined by electron microscopy. In situ DNA hybridization, using a canine oral papillomavirus probe, localized papillomavirus DNA in canine oral papillomas, but not in canine cutaneous squamous or inverted papillomas, suggesting that a different papillomavirus type was present in the latter lesions. Although these lesions resembled intracutaneous cornifying epitheliomas (keratoacanthomas), they appear to be a distinct lesion, probably with a different etiology.     

Real-time PCR analysis of p53 mRNA levels in tissues of whitefish (Coregonus lavaretus) exposed to benzo[a]pyrene

We developed a real-time PCR assay for measuring relative quantities (RQ) of p53 tumor suppressor mRNA in the whitefish (Coregonus lavaretus, Salmonidae, Teleostei). Real-time PCR primers for the p53 gene were designed from a region that was found to be conserved among salmonid p53 genes. To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a putative p53-inducer. Two groups of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were either given an intraperitoneal injection (10 mg x kg(-1)) of B[a]P in corn oil (2 mg B[a]P ml(-1) corn oil) or corn oil alone (Control). After treatment (48 h, 7 degrees C), two random fish from each group were anesthetized and the liver, head kidney and brain were collected for mRNA isolation and analysis. In the control fish, relative quantification analysis based on the p53 mRNA levels in liver (RQ=1.00) showed higher basal levels of p53 mRNA in the head kidney (RQ= 1.69), and lower in the brain (RQ=0.41). In all three tissues sampled, p53 mRNA was affected by treatment with B[a]P. Liver tissue showed the greatest induction (RQ=1.53) from base levels (RQ=1.00), followed by brain (RQ=1.36), and head kidney (RQ=1.23). These results confirm that p53 mRNA is generally present at lower levels in differentiated tissues (liver and brain) than in those tissues with cell lines (head kidney), and demonstrate that p53 is moderately inducible by B[a]P in the whitefish. The approach presented here has the advantage of providing rapid and accurate measures of p53 induction in various tissues of fish responding to PAH contaminant exposure.